Abstract It is now clear that epigenetic mechanisms control gene transcription in area CA1 of the hippocampus that are necessary for proper memory formation in young and mature adulthood. For example, we have discovered that epigenetic mechanisms thought to be static after development, remain active in postmitotic neurons in adulthood and very much responsive to environmental experiences. Specifically, histone lysine methylation (HKM) mechanisms have been identified as a crucial transcriptional mechanism in area CA1 subserving the process of associative memory formation. Surprisingly little is known about how environmental influences like aging and stress experiences contribute to memory decline with age. Our long-term goal is to identify HKM modifications induced by stress to impact memory formation with aging. This has tremendous implications with regard to long- lasting epigenetic effects, which is rarely observed in the field of cognitive neuroepigenetics. For this grant proposal, we will focus on the histone lysine methyltransferase, G9a that mediates histone H3-lysine 9 dimethylation (H3K9me2) marks in area CA1 neurons. With this in mind, our preliminary results demonstrate that severe stress increased G9a-H3K9me2 activity in area CA1 of young adult rats that was sustained with aging. Furthermore, H3K9me2 hypermethylation strongly correlated with memory decline in young adults and in aged adults. Preliminary results also demonstrate that inhibiting G9a activity in the hippocampus reverses stress- induced memory impairments. Based on these preliminary results, we plan to rigorously investigate the beneficial effects of manipulating G9a-H3K9me2 activity in CA1 following stress and determine effects on age-related memory decline. To gain further mechanistic insight into stress-related G9a-H3K9me2 mediated gene transcription, we will use state-of-the-art approaches such as siRNA and CRISPR-dCas9-VP64 technology to directly target G9a in our animal model system. Our central hypotheses are 1) stress abnormally increases G9a- H3K9me2- mediated silencing of hippocampal genes in young adult rats similar to what is observed in normal aging adults, 2) stress-mediated G9a-H3K9me2 hypermethylation will correlate with aged-impaired but not aged- unimpaired animals, and 3) stress experienced in young adults results in latent effects on H3K9me2 hypermethylation with aging that can be overcome by G9a inhibition. Our Specific Aims are as follows: Specific Aim 1: Stress interferes with memory formation by increasing G9a-H3K9me2 activity in the hippocampus of young adults; Specific Aim 2: Stress has differing effects on G9a-H3K9me2 in the hippocampus of aged- impaired versus aged-unimpaired animals; and Specific Aim 3: Stress experience increases G9a-H3K9me2 activity in the hippocampus of young adults contributing to memory decline with age. Collectively, this proposal will identify long-lasting epigenetic mechanisms involved in stress-related memory decline with age. Moreover, we hope to add to our understanding of stress-induced effects on memory decline as the brain ages.